Human monoclonal antibodies broadly protective against influenza b virus and methods of using the same

ABSTRACT

Materials and methods are provided for treating influenza B infections in humans. Anti-human influenza virus monoclonal antibodies and antigen-binding fragments thereof having a neutralization activity against a human influenza B virus are provided. Methods for producing anti-human influenza B virus monoclonal antibodies are also provided. The antibodies and antigen-binding fragments thereof can be effective against a wide range of influenza B viral strains. Methods of inhibiting or treating a human influenza B infection are provided. The anti-influenza B therapeutics can also be used to manufacture medicaments effective against influenza B infections, to detect human influenza B in a human subject, for use in pharmaceutical compositions, and for use in kits for at least one of the prevention, the treatment, and the detection of human influenza B in a human subject.

RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 61/592,657, filed on Jan. 31, 2012, which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

The subject matter described herein was supported, at least in part, bythe Japan Science and Technology Agency/Japan International CooperationAgency, Science and Technology Research Partnership for SustainableDevelopment (JST/JICA, SATREPS); and a Grant-in-Aid for Young Scientists(B) from the Japan Society for the Promotion of Science to Mayo Yasugi.

TECHNICAL FIELD

The present invention relates to materials and methods for the treatmentof influenza B viral infections in humans.

BACKGROUND ART

The ability of influenza virus to evade immune surveillance throughrapid genetic drift and reassortment means that it remains a continuouspublic health threat. During annual epidemics five to fifteen percent ofthe worldwide population are typically infected, resulting in threemillion to five million cases of severe illness and between 250,000 to500,000 deaths per year (Lambert et al., The New England Journal ofMedicine 363:2036-2044; WHO, Influenza (Seasonal) Fact Sheet No. 211).Influenza B virus, like H1 and H3 subtypes of influenza A virus, hascaused epidemics in humans (WHO, Influence (Seasonal) Fact Sheet No.211). In contrast to influenza A, influenza B virus is found almostexclusively in humans and has a much slower mutational rate than thatobserved for influenza A virus (Carrat et al., Vaccine 25:6852-6862;Nobusawa et al., Journal of Virology 80:3675-3678; Webster et al, TheJournal of General Virology 54:243-251). However, co-circulation of twophylogenetically and antigenically distinct lineages, represented by theB/Yamagata/16/88 and B/Victoria/2/87, has caused antigenic variationthrough genetic reassortment and antigenic drift from cumulativemutations, leading to annual endemics (Hay et al., PhilosophicalTransactions of the Royal Society of London 356:1861-1870; Lin et al.,Virus Research 103:47-52).

The development of vaccines producing broadly reactive antibodies andtherapeutic strategies using human monoclonal antibodies (HuMAbs) withglobal reactivity has recently been gathering great interest.Neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza)have been widely used for the treatment of influenza viral infection.However, they have limited efficacy when administered more than 48 hoursafter the onset of illness (Aoki et al., The Journal of AntimicrobialChemotherapy 51: 123-129), and widespread use has resulted in theemergence of resistant viral strains (Kiso et al, Lancet 364:759-765;Lowen et al., Infectious Disorders Drug Targets 7:318-328; Reece,Journal of Medical Virology 79:1577-1586). Therefore, new therapeuticstrategies that provide potent and broadly cross-protective hostimmunity are a global public health priority. Thus, the development ofnovel antibody-based therapies is of great interest (Marasco et al.,Nature Biotechnology 25:1421-1434).

Several human monoclonal antibodies (HuMAbs) with broad neutralizingactivities were identified against the hemagglutinin (HA) protein ininfluenza A viruses, including C6261 and F10, which react with group 1viruses (Ekiert et al., Science 324:246-251; Sui et al., NatureStructural & Molecular Biology 16:265-273), and CR8020, whichneutralizes group 2 viruses (Ekiert et al., Science 333:843-850).Another HuMAb, FI6v3, which neutralized both group 1 and group 2influenza A viruses, was isolated in 2011 (Corti et al., Science 333:850-856). Although influenza B virus has a much slower mutational ratethan that observed for influenza A virus like H1 and H3 subtypes, it hasannually caused epidemics in humans (NPL1: Hay et al., PhilosophicalTransactions of the Royal Society of London 256:1861-1870; NPL2: Lin etal., Virus Research 103:47-52). For influenza B virus, however, broadlyneutralizing HuMAbs, CR8033, CR8071 and CR9114, have firstly reported onSeptember 2012 (NPL3: Dreyfus et al., Science 337: 1343-1348).Accordingly, a need exists for broadly neutralizing HuMAb against theinfluenza B virus.

CITATION LIST Non Patent Literature

-   [NPL 1] Hay et al., Philos Trans R Soc Lond B Biol Sci. 2001 Dec.    29; 356(1416): 1861-1870.-   [NPL 2] Lin et al., Virus Res. 2004 July; 103(1-2):47-52.-   [NPL 3] Dreyfus et al., Science. 2012 Sep. 14; 337(6100):1343-8.    doi: 10.1126/science.1222908. Epub 2012 Aug. 9.

SUMMARY OF INVENTION Technical Problem

In the current study, three HuMAbs were prepared that broadly reacted tothe HA protein in influenza B virus. Three HuMAbs designated 5A7, 3A2,and 10C4 against influenza B virus were prepared using peripherallymphocytes from vaccinated volunteers. In vitro, HuMAb 5A7 broadlyneutralized influenza B strains isolated from 1985 to 2006, whereas 3A2and 10C4 reacted to the Yamagata lineage only. Epitope mapping revealedthat 3A2 and 10C4 recognized the 190-helix region near the receptorbinding site in the hemagglutinin (HA) protein. Amino acid residues ofthe 190-helix readily mutate. 5A7 recognized amino acid positions 315 to324 near the C terminal of HA 1, a highly conserved region in influenzaB viruses. Moreover, 5A7 showed therapeutic efficacy in mice even ifHuMAb was injected 72 hours post-infection. HuMAb 5A7 synthesized fromfull-length variable gene-transfected CHO-K1 cells also showedneutralizing activity against influenza B viruses. These resultsindicate that the antibodies of the invention, including 5A7, can beused as therapeutics against influenza B virus.

It is therefore a feature of the present invention to providetherapeutics including antibodies and antigen-binding fragments thereofcapable of preventing, inhibiting, and treating an influenza Binfection.

Another feature of the present invention is to provide methods forproducing such therapeutics.

A further feature of the present invention is to provide therapeuticseffective against a wide range of influenza B viral strains.

Additional features and advantages of the present invention will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of thepresent invention. The objectives and other advantages of the presentinvention will be realized and attained by means of the elements andcombinations particularly pointed out in the description and appendedclaims.

Solution to Problem

To achieve these and other advantages, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the present invention relates to an anti-human influenza virusmonoclonal antibody or an antigen-binding fragment thereof having aneutralization activity against a human influenza B virus, wherein themonoclonal antibody can include a human monoclonal antibody and/or ahumanized monoclonal antibody.

The present invention provides a method for producing an anti-humaninfluenza B virus monoclonal antibody. The method can include producinga hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from ahuman being, for example a patient and/or a vaccine in an influenza Bvirus infection with a fusion partner cell capable of efficient cellfusion. The method can also include obtaining an anti-human influenzavirus monoclonal antibody from the hybridoma. The influenza B virus inthe method can include at least one of a B/Florida/4/2006 strain, aB/Shanghai/361/2002/strain, a B/Johannesburg/5/1999 strain, aB/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04strain, a B/Shandong/7/1997 strain, and B/Victoria/2/1987 strain. Thefusion partner cell is a SPYMEG cell. Thus, an anti-human influenzavirus monoclonal antibody produced by the method is further provided.

The present invention provides a method of inhibiting or treating ahuman influenza B infection in a human subject including administering atherapeutically effective amount of the anti-human influenza B virushuman antibody or antigen-binding fragment thereof of the invention tothe human subject. The method can further include diagnosing the patientwith an influenza B infection. The method can further include monitoringfor a decrease in at least one symptom of an influenza B infection.

The present invention provides use of an anti-human influenza B virusmonoclonal antibody or antigen-binding fragment thereof of the presentinvention to manufacture a medicament for inhibiting or treating a humaninfluenza B infection in a human subject. The present invention alsoprovides a method of detecting human influenza B in a human subjectincluding contacting a sample from the human subject with an anti-humaninfluenza B virus monoclonal antibody or antigen-binding fragmentthereof of the invention. The present invention further provides apharmaceutical composition containing an anti-human influenza virusmonoclonal antibody or antigen-binding fragment thereof of the presentinvention and a pharmacologically acceptable carrier. The presentinvention still further provides a kit for at least one of theprevention, the treatment, and the detection of human influenza B in ahuman subject containing an anti-human influenza virus monoclonalantibody or antigen-binding fragment thereof of the present invention.The kit can include the pharmaceutical composition and/or one or moreadditional influenza B or other antagonists.

The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate some of the embodiments of thepresent invention and together with the description, serve to explainthe principles of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the sequences of escape mutants selected by the incubationof B/Florida/4/2006 with HuMAbs. The amino acid sequences of the HAprotein in the escape mutants are compared with the originalB/Florida/4/2006. Asterisks indicate the amino acid residues differentbetween the original virus and the escape mutants.

FIG. 2 shows a series of truncated forms of HA (a to f) in an expressionplasmid, which were prepared as depicted in the diagram. Transfected293T cells were subjected to Western blotting with 5A7. The serum from amouse infected with B/Ibaraki/2/1985 was used as a control (Ms serum).

FIG. 3 shows expression plasmids bearing chimeric HA protein preparedwith B/Shanghai/361/2002 (Sh/02) and B/Florida/4/2006 (Flo/06). 293Tcells expressing the chimeric protein were subjected to animmunofluorescence assay (IFA) with 3A2 (left panels). White barsrepresent the amino acid sequence in Sh/02, and black bars represent theamino acid sequence in Flo/06. The different amino acid residues in theHA protein from each of the two viral strains are shown in the top andbottom bars.

FIG. 4 shows an epitope map of the HuMAbs in three-dimensional structureof the HA protein.

FIG. 5 shows the therapeutic efficacy of 5A7 in mice. Mice were treatedintraperitoneally with HuMAb at 5, 10, or 15 mg/kg or with PBS at 4hours post-challenge with a lethal dose (2.5×10⁴ FFU/mouse) ofmouse-adapted B/Ibaraki/2/1985. Survival and body weight were checkeddaily. Each group consists of five mice. Body weight is shown as themean+ or −SEM of five mice.

FIG. 6 shows the therapeutic efficacy of 5A7 in mice. Mice were treatedintraperitoneally with HuMAb at 5, 10, or 15 mg/kg or with PBS at 4hours post-challenge with a lethal dose (2.5×10⁴ FFU/mouse) ofmouse-adapted B/Ibaraki/2/1985. Survival and body weight were checkeddaily. Each group consists of five mice. Body weight is shown as themean+ or −SEM of five mice.

FIG. 7 shows mice treated with 5A7 at 10 mg/kg or with PBS at 4 hourspost-challenge with 2.5×10⁴ FFU/mouse mouse-adapted B/Ibaraki/2/1985(left panel) and 5.0×10³FFU/mouse B/Florida/4/2006 (right panel). Thetiters in lungs were calculated at 3 and 6 days post-infection. Eachgroup consists of three mice. Black bars are mean values. Asterisksdenote P<0.05 compared to the PBS-treated group.

FIG. 8 shows that mice were given 10 mg/kg HuMAb or PBS at 4, 24, 48, or72 hours post-infection (hpi) with mouse-adapted B/Ibaraki/2/1985(2.5×10⁴ FFU/mouse). Survival and body weight were checked daily. Eachgroup consists of ten mice. Body weight is shown as the mean+ or −SEM often mice.

FIG. 9 shows that mice were given 10 mg/kg HuMAb or PBS at 4, 24, 48, or72 hours post-infection (hpi) with mouse-adapted B/Ibaraki/2/1985(2.5×10⁴ FFU/mouse). Survival and body weight were checked daily. Eachgroup consists of ten mice. Body weight is shown as the mean+ or −SEM often mice.

FIG. 10 shows an in vitro neutralization assay using synthesized 5A7from CHO-K1 cells. The infectivity of B/Florida/4/2006 andB/Malaysia/2506/2004 were measured in the presence of synthesized 5A7from CHO-K1 (5A7/CHO; solid lines) and compared with infectivity in thepresence of 5A7 from hybridoma supernatant (5A7/hybridoma; dashedlines). HuMAbs (100 mcg/ml (microgram/ml)) were serially dilutedfour-fold.

FIG. 11 shows sequences of the V_(H) and V_(L) region of the threeHuMAbs. These three HuMAbs were derived from different germ lines exceptD region V_(H) of 3A2 and 10C4.

FIG. 12 shows sequences of the V_(H) and V_(L) region of the threeHuMAbs. These three HuMAbs were derived from different germ lines exceptD region V_(H) of 3A2 and 10C4.

FIG. 13 shows sequences of the V_(H) and V_(L) region of the threeHuMAbs. These three HuMAbs were derived from different germ lines exceptD region V_(H) of 3A2 and 10C4. The accompanying drawings, which areincorporated into and constitute a part of the specification, illustratespecific features/embodiments of the present invention, and taken inconjunction with the detailed description, serve to explain theprinciples of the present invention. It is to be understood that boththe foregoing general description and the following detailed descriptionare exemplary and explanatory only and are intended to provide a furtherexplanation of the present invention, as claimed.

DESCRIPTION OF EMBODIMENTS Detailed Description of the Present Invention

The present invention provides an anti-human influenza virus monoclonalantibody or an antigen-binding fragment thereof having a neutralizationactivity against a human influenza B virus, wherein the monoclonalantibody includes a human monoclonal antibody and/or a humanizedmonoclonal antibody. The monoclonal antibody or antigen-binding fragmentthereof can have a neutralization activity against at least aB/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, and aB/Mie/1/1993 strain. The monoclonal antibody or antigen-binding fragmentthereof can have a neutralization activity against at least aB/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and a B/Victoria/2/1987 strain. The anti-human influenza B virusmonoclonal antibody or antigen-binding fragment thereof can include anIgG, a Fab, a Fab′, a F(ab′)2, a scFv, a dsFv, or any combinationthereof.

The anti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof can include at least one heavy chain variable regionand/or at least one light chain variable region. The heavy chainvariable region can include at least one of a first complementaritydetermining region (CDR1), a second complementarity determining region(CDR2), and a third complementarity determining region (CDR3). The CDR1of the heavy chain variable region can have a first amino acid sequenceincluding SEQ ID NO: 1, 7, or 13. The CDR2 of the heavy chain variableregion can have a second amino acid sequence including SEQ ID NO: 2, 8,or 14. The CDR3 of the heavy chain variable region can have a thirdamino acid sequence including SEQ ID NO: 3, 9, or 15. The light chainvariable region can also include at least one of a first complementaritydetermining region (CDR1), a second complementarity determining region(CDR2), and a third complementarity determining region (CDR3). The CDR1of the light chain variable region can have a fourth amino acid sequenceincluding SEQ ID NO: 4, 10, or 16. The CDR2 of the light chain variableregion can have a fifth amino acid sequence including SEQ ID NO: 5, 11,or 17. The CDR3 of the light chain variable region can have a sixthamino acid sequence including SEQ ID NO: 6, 12, or 18.

The anti-human influenza virus monoclonal antibody or antigen-bindingfragment can have the first amino acid sequence include SEQ ID NO: 1,the second amino acid sequence include SEQ ID NO: 2, the third aminoacid sequence include SEQ ID NO: 3, the fourth amino acid sequenceinclude SEQ ID NO: 4, the fifth amino acid sequence include SEQ ID NO:5, and the sixth amino acid sequence include SEQ ID NO: 6. For example,the anti-human influenza virus monoclonal antibody can include antibody5A7. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof can have the first amino acid sequenceinclude SEQ ID NO: 7, the second amino acid sequence include SEQ ID NO:8, the third amino acid sequence include SEQ ID NO: 9, the fourth aminoacid sequence include SEQ ID NO: 10, the fifth amino acid sequenceinclude SEQ ID NO: 11 and the sixth amino acid sequence include SEQ IDNO: 12. For example, the anti-human influenza virus monoclonal antibodycan include antibody 3A2. The anti-human influenza virus monoclonalantibody or antigen-binding fragment thereof can have the first aminoacid sequence include SEQ ID NO: 13, the second amino acid sequenceinclude SEQ ID NO: 14, the third amino acid sequence include SEQ ID NO:15, the fourth amino acid sequence include SEQ ID NO: 16, the fifthamino acid sequence include SEQ ID NO: 17, and the sixth amino acidsequence include SEQ ID NO: 18. For example, the anti-human influenzavirus monoclonal antibody can include antibody 10C4.

The monoclonal antibody can be produced by a hybridoma made by fusing aperipheral blood mononuclear cell (PBMC) from a human being, for examplea patient and/or a vaccine having an influenza B virus infection with afusion partner cell capable of efficient cell fusion. The influenza Bvirus of the infection can include at least one of a B/Florida/4/2006strain, a B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, aB/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04strain, a B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain. Thefusion partner cell can be a SPYMEG cell.

Accordingly, the present invention provides a method for producing ananti-human influenza B virus monoclonal antibody. The method can includeproducing a hybridoma by fusing a peripheral blood mononuclear cell(PBMC) from a human being, for example a patient and/or a vaccine in aninfluenza B virus infection with a fusion partner cell capable ofefficient cell fusion. The method can also include obtaining ananti-human influenza virus monoclonal antibody from the hybridoma. Theinfluenza B virus in the method can include at least one of aB/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and B/Victoria/2/1987 strain. The fusion partner cell can be aSPYMEG cell. Thus, an anti-human influenza virus monoclonal antibodyproduced by the method is further provided. The monoclonal antibody caninclude, for example, a human monoclonal antibody and/or a humanizedmonoclonal antibody.

Anti-influenza antibodies and polypeptides containing antigen bindingfragments thereof are provided as well as methods, uses, compositions,and kits employing the same. A method of forming an antibody specific toan influenza or a polypeptide or a fragment thereof is provided. Such amethod can contain providing a nucleic acid encoding a influenza antigenpolypeptide or a polypeptide containing an immunologically specificepitope thereof; expressing the polypeptide containing the antigen aminoacid sequence or a polypeptide containing an immunologically specificepitope thereof from the isolated nucleic acid; and generating anantibody specific to the polypeptide obtained or a polypeptidecontaining an antigen binding fragment thereof. An antibody orpolypeptide containing an antigen binding fragment thereof produced bythe aforementioned method is provided. An isolated antibody or isolatedpolypeptide containing an antigen binding fragment thereof thatspecifically binds an influenza antigen is provided. Such an antibodycan be generated using any acceptable method(s) known in the art. Theantibodies as well as kits, methods, and/or other aspects of the presentinvention employing antibodies can include one or more of the following:a polyclonal antibody, a monoclonal antibody, a chimeric antibody, asingle-chain antibody, a monovalent antibody, a diabody, and/or ahumanized antibody.

Naturally occurring antibody structural units typically contain atetramer. Each such tetramer can be composed of two identical pairs ofpolypeptide chains, each pair having one full-length light” (forexample, about 25 kDa) and one full-length “heavy” chain (for example,about 50-70 kDa). The amino-terminal portion of each chain typicallyincludes a variable region of about 100 to 110 or more amino acids thattypically is responsible for antigen recognition. The carboxy-terminalportion of each chain typically defines a constant region that may beresponsible for effector function. Human light chains are typicallyclassified as kappa and lambda light chains. Heavy chains are typicallyclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to, IgG1, IgG2, IgG3, andIgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2.IgA is similarly subdivided into subclasses including, but not limitedto, IgA1 and IgA2. In light and heavy chains, the variable and constantregions can be joined by a “J” region of about 12 or more amino acids,with the heavy chain also including a “D” region of about 10 or moreamino acids. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N. Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair typically form the antigen binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions orCDRs. The CDRs from the two chains of each pair typically are aligned bythe framework regions, which can enable binding to a specific epitope.From N-terminal to C-terminal, both light and heavy chain variableregions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989).

“Antibody fragments” include a portion of an intact antibody, such asthe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab 1, F(ab′)2, and Fv fragments;diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, a designation reflecting the ability to crystallizereadily. Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.“Fv” is an antibody fragment which contains a completeantigen-recognition and -binding site. This region includes a dimer ofone heavy- and one light-chain variable domain in tight, non-covalentassociation. A single variable domain (or half of an Fv containing onlythree CDRs specific for an antigen) can recognize and bind an antigen.“Single-chain Fv” or “sFv” antibody fragments include the V_(H) andV_(L) domains of the antibody, wherein these domains are present in asingle polypeptide chain. The Fv polypeptide can further contain apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

Antibodies can be used as probes, therapeutic treatments and other uses.Antibodies can be made by injecting mice, rabbits, goats, or otheranimals with the translated product or synthetic peptide fragmentsthereof. These antibodies are useful in diagnostic assays or as anactive ingredient in a pharmaceutical composition.

The antibody or polypeptide administered can be conjugated to afunctional agent to form an immunoconguate. The functional agent can bea cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate), an antibiotic, a nucleolytic enzyme, or any combinationthereof. Chemotherapeutic agents can be used in the generation ofimmunoconjugates, e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents, enzymes,and/or fragments thereof, such as nucleolytic enzymes, antibiotics, andtoxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof, and the various antitumor or anticancer agentsdisclosed below. Enzymatically active toxins and fragments thereof thatcan be used include, for example, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricotheeenes.Any appropriate radionucleotide or radioactive agent known in the art orare otherwise available can be used to produce radioconjugatedantibodies.

Conjugates of the antibody and cytotoxic agent can be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP); iminothiolane(IT); bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL); active esters (such as disuccinimidyl suberate);aldehydes (such as glutareldehyde); bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine); bis-diazonium derivatives (such asbis-(p-diazo-niumbenzoyl)-ethylenediamine); diisocyanates (such astolyene 2,6-diisocyanate); bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene); maleimidocaproyl (MC);valine-citrulline, dipeptide site in protease cleavable linker (VC);2-amino-5-ureido pentanoic acid PAB=p-aminobenzylcarbamoyl (“selfimmolative” portion of linker) (Citrulene); N-methyl-valine citrullinewhere the linker peptide bond has been modified to prevent its cleavageby cathepsin B (Me); maleimidocaproyl-polyethylene glycol, attached toantibody cysteines; N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP);and N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate(SMCC). For example, a ricin immunotoxin can be prepared as described inVitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacctic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody, see WO 94/11026. The antibody can beconjugated to a “receptor” (such as streptavidin) for utilization intumor pre-targeting wherein the antibody-receptor conjugate isadministered to the subject, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., aradionucleotide).

The antibodies of the present invention can be coupled directly orindirectly to a detectable marker by techniques well known in the art. Adetectable marker is an agent detectable, for example, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Usefuldetectable markers include, but are not limited to, fluorescent dyes,chemiluminescent compounds, radioisotopes, electron-dense reagents,enzymes, colored particles, biotin, or dioxigenin. A detectable markeroften generates a measurable signal, such as radioactivity, fluorescentlight, color, or enzyme activity. Antibodies conjugated to detectableagents may be used for diagnostic or therapeutic purposes. Examples ofdetectable agents include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance can be coupled or conjugated either directly to theantibody or indirectly, through an intermediate such as, for example, alinker known in the art, using techniques known in the art. See, e.g.,U.S. Pat. No. 4,741,900, describing the conjugation of metal ions toantibodies for diagnostic use. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, andacetyl-cholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, and phycoerythrin; an example of a luminescent materialincludes luminol; examples of bioluminescent materials includeluciferin, and aequorin.

Antibodies useful in practicing the present invention can be prepared inlaboratory animals or by recombinant DNA techniques using the followingmethods. Polyclonal antibodies can be raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the gene productmolecule or fragment thereof in combination with an adjuvant such asFreund's adjuvant (complete or incomplete). To enhance immuno-genicity,it can be useful to first conjugate the gene product molecule or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl, etc. Alternatively,immunogenic conjugates can be produced recombinantly as fusion proteins.

Animals can be immunized against the immunogenic conjugates orderivatives (such as a fragment containing the target amino acidsequence) by combining about 1 mg or about 1 microgram of conjugate (forrabbits or mice, respectively) with about 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites.Approximately 7 to 14 days later, animals are bled and the serum isassayed for antibody titer. Animals are boosted with antigen repeatedlyuntil the titer plateaus. The animal can be boosted with the samemolecule or fragment thereof as was used for the initial immunization,but conjugated to a different protein and/or through a differentcross-linking agent. In addition, aggregating agents such as alum can beused in the injections to enhance the immune response.

The antibody administered can include a chimeric antibody. The antibodyadministered can include a humanized antibody. The antibody administeredcan include a completely humanized antibody. The antibodies can behumanized or partially humanized. Non-human antibodies can be humanizedusing any applicable method known in the art. A humanized antibody canbe produced using a transgenic animal whose immune system has beenpartly or fully humanized. Any antibody or fragment thereof of thepresent invention can be partially or fully humanized. Chimericantibodies can be produced using any known technique in the art. See,e.g., U.S. Pat. Nos. 5,169,939; 5,750,078; 6,020,153; 6,420,113;6,423,511; 6,632,927; and 6,800,738.

The antibody administered can include a monoclonal antibody, that is,the anti-influenza antibodies of the present invention that can bemonoclonal antibodies. Monoclonal antibodies can be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes can be immunized in vitro. Monoclonalantibodies can be screened as are described, for example, in Harlow &Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, NewYork (1988); Goding, Monoclonal Antibodies, Principles and Practice (2ded.) Academic Press, New York (1986). Monoclonal antibodies can betested for specific immunoreactivity with a translated product and lackof immunoreactivity to the corresponding prototypical gene product.

Monoclonal antibodies can be prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g., by fusion with myeloma cells. The clones are then screened forthose expressing the desired antibody. The monoclonal antibodypreferably does not cross-react with other gene products. After thedesired hybridoma cells are identified, the clones can be subcloned bylimiting dilution procedures and grown by standard methods. Suitableculture media for this purpose include, for example, Dulbecco's ModifiedEagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cellscan be grown in vivo as ascites in a mammal. The monoclonal antibodiessecreted by the subclones can be isolated or purified from the culturemedium or ascites fluid by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the present invention can be readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the present invention can serve as a preferred source of suchDNA. Once isolated, the DNA can be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also can be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequencesor by covalently joining to the immunoglobulin coding sequence all orpart of the coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the present invention, or can be substitutedfor the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody. Preparation ofantibodies using recombinant DNA methods such as the phagemid displaymethod, can be accomplished using commercially available kits, as forexample, the Recombinant Phagemid Antibody System available fromPharmacia (Uppsala, Sweden), or the SurfZAP™ phage display system(Stratagene Inc., La Jolla, Calif.).

Also included in the present invention are hybridoma cell lines,transformed B cell lines, and host cells that produce the monoclonalantibodies of the present invention; the progeny or derivatives of thesehybridomas, transformed B cell lines, and host cells; and equivalent orsimilar hybridomas, transformed B cell lines, and host cells.

The antibodies can be diabodies. The term “diabodies’ refers to smallantibody fragments with two antigen-binding sites, which fragmentsinclude a heavy-chain variable domain (V_(H)) connected to a light-chainvariable domain (V_(L)) in the same polypeptide chain (Vn-V_(L)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains can be forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, for example, EP 404,097;WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993).

The antibody administered can include a single-chain antibody. Theantibodies can be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain can be truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

The antibodies can be bispecific. Bispecific antibodies thatspecifically bind to one protein and that specifically bind to otherantigens relevant to pathology and/or treatment are produced, isolated,and tested using standard procedures that have been described in theliterature. [See, e.g., Pluckthun & Pack, Immunotechnology, 3:83-105(1997); Carter, et al., J. Hematotherapy, 4:463-470 (1995); Renner &Pfreundschuh, Immunological Reviews, 1995, No. 145, pp. 179-209;Pfreundschuh U.S. Pat. No. 5,643,759; Segal, et al., J. Hematotherapy,4:377-382 (1995); Segal, et al., Immunobiology, 185:390-402 (1992); andBolhuis, et al., Cancer Immunol. Immunother., 34:1-8 (1991)].

The antibodies disclosed herein can be formulated as immunoliposomes.Liposomes containing the antibody are prepared by methods known in theart. such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition containingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes can be extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257:286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al, J. National Cancer Inst.,81(19): 1484 (1989).

The influenza antagonist can include an aptamer that binds thehemagglutinin (HA) protein of an influenza B virus. The aptamer can bindHA in the same location/epitope as the antibodies described hereinand/or to other locations/epitopes. The aptamer can contain one or moreof a nucleic acid, a RNA, a DNA, and an amino acid. Aptamers can beselected and produced using any suitable technique or protocol. Forexample, oligonucleotide libraries with variable regions ranging from 18to 50 nucleotides in length can be used as templates for run-offtranscription reactions to generate random pools of RNA aptamers. Thisaptamer pool can then be exposed to unconjugated matrix to removenon-specific interacting species. The remaining pool is then incubatedwith an immobilized target. The majority of aptamer species in this poolcan have low affinity, for the target can be washed away leaving asmaller, more specific pool bound to the matrix. This pool can then beeluted, precipitated, reverse transcribed, and used as a template forrun-off transcription. After five rounds of selection, aliquots can beremoved that are cloned and sequenced. Selection can be continued untilsimilar sequences are reproducibly recovered.

Aptamer production can be performed using a bead-based selection system.In this process, a library of beads is generated in which each bead iscoated with a population of aptamers with identical sequences composedof natural and modified nucleotides. This bead library, which cancontain greater than 100,000,000 unique sequences, can be incubated witha peptide that corresponds to hemagglutinin (HA) protein, or a portionthereof, e.g., an extracellular domain, that is conjugated with a tagsuch as a fluorescent dye. After washing, beads that demonstrate thehighest binding affinity can be isolated and aptamer sequences can bedetermined for subsequent synthesis.

The present invention provides a method of inhibiting or treating ahuman influenza B infection in a human subject including administering atherapeutically effective amount of the anti-human influenza B virusmonoclonal antibody or antigen-binding fragment thereof of the inventionto the human subject. The method can further include diagnosing thepatient with an influenza B infection. Anti-influenza antibodies orantigen-binding fragment thereof of the present invention can beadministered to a subject before, during, and/or after diagnosing thepatient as having an influenza infection.

The method can further include monitoring for a decrease in at least onesymptom of an influenza B infection. For example, the at least onesymptom can include fever, headache, fatigue, chills, malaise, myalgia,arthralgia, nasal congestion, sore throat, cough, respiratory distress,stomach pain, or any combination thereof. The anti-human influenza virusmonoclonal antibody or antigen-binding administered in combination withone or more additional therapies directed to influenza B and/or otherinfluenzas such as influenza A and/or influenza C. The combination canact synergistically to inhibit or treat the influenza B infection. Theone or more additional therapies can include, for example, aneuraminidase inhibitor, a hemagglutinin inhibitor, an anti-inflammatoryagent, or any combination thereof. The neuraminidase inhibitor caninclude, for example, zanamivir, oseltamivir, peramivir, laninamivir,any pharmaceutically acceptable salt thereof, or any combinationthereof.

In accordance with the present invention, two or more influenzaantagonists can be administered. At least one of the influenzaantagonists can include an influenza B antagonist. The at least oneinfluenza B antagonist can be combined with one or more influenza Aantagonists and/or one or more influenza C antagonists. At least oneinfluenza antagonist can be administered in combination with one or moreadditional therapies directed against an influenza viral infection. Theadministration of two or more therapies, including one or more influenzaantagonists, can be simultaneous, sequential, or in combination.Accordingly, when two or more therapies are administered, they need notbe administered simultaneously or in the same way or in the same dose.When administered simultaneously, the two or more therapies can beadministered in the same composition or in different compositions. Thetwo or more therapies can be administered using the same route ofadministration or different routes of administration. When administeredat different times, the therapies can be administered before or aftereach other. Administration order of the two or more therapies can bealternated. The respective doses of the one or more therapies can bevaried over time. The type of one or more therapy can be varied overtime. When administered at separate times, the separation of the two ormore administrations can be any time period. If administered multipletimes, the length of the time period can vary. The separation betweenadministration of the two or more two or more therapies can be 0seconds, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5minutes, 10 minutes, 15 minutes, 20 minutes, 30, minutes, 45 minutes, 1hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7.5hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 1.5days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks,3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, three months, six months,1 year or longer.

Two or more influenza antagonists can act synergistically to treat orreduce an influenza infection or a symptom of the same, for example,fever. An influenza antagonist can be one or more anti-influenzaantibody alone or in combination with one or more other influenzaantagonist, for example, a small drug pharmaceutical, or otheranti-influenza therapy. Two or more anti-influenza antibodies, or atleast one anti-influenza antibody and one or more additional therapiescan act synergistically to treat or reduce an influenza B viralinfection. Two or more therapies, including one or more anti-influenzaantibody, can be administered in synergistic amounts. Accordingly, theadministration of two or more therapies can have a synergistic effect onthe decrease in one or more symptoms of an influenza infection, whetheradministered simultaneously, sequentially, or in any combination. Afirst therapy can increase the efficacy of a second therapy greater thanif second therapy was employed alone, or a second therapy increases theefficacy of a first therapy, or both. The effect of administering two ormore therapies can be such that the effect on decreasing one or moresymptoms of an influenza infection is greater than the additive effectof each being administered alone. When given in synergistic amounts, onetherapy can enhance the efficacy of one or more other therapy on thedecrease in one or more symptoms of an influenza infection, even if theamount of one or more therapy alone would have no substantial effect onone or more symptom of an influenza infection. Measurements andcalculations of synergism can be performed as described in Teicher,“Assays for In Vitro and In Vivo Synergy,” in Methods in MolecularMedicine, vol. 85: Novel Anticancer Drug Protocols, pp. 297-321 (2003)and/or by calculating the combination index (CI) using CalcuSynsoftware.

The present invention provides use of an anti-human influenza B virusmonoclonal antibody or antigen-binding fragment thereof of the presentinvention to manufacture a medicament for inhibiting or treating a humaninfluenza B infection in a human subject. The present invention alsoprovides a method of detecting human influenza B in a human subject. Themethod can include contacting a sample from the human subject with ananti-human influenza B virus human antibody or antigen-binding fragmentthereof of the invention. The method can further include detecting thepresence or absence of a human influenza B virus in the human subjectbased on whether the antibody binds a hemagglutinin (HA) protein of thehuman influenza B virus. The present invention further provides apharmaceutical composition containing an anti-human influenza virushuman antibody or antigen-binding fragment thereof of the presentinvention and a pharmacologically acceptable carrier. The presentinvention still further provides a kit for at least one of theprevention, the treatment, and the detection of human influenza B in ahuman subject containing an anti-human influenza virus monoclonalantibody or antigen-binding fragment thereof of the present invention.The kit can include the pharmaceutical composition and/or one or moreadditional anti-influenza B or other antagonists.

Exact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition. [See, e.g.,Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1p. I.] The attending physician can determine when to terminate,interrupt, or adjust administration due to toxicity, or to organdysfunctions. Conversely, the attending physician can also adjusttreatment to higher levels if the clinical response were not adequate,precluding toxicity. The magnitude of an administrated dose in themanagement of disorder of interest will vary with the severity of thedisorder to be treated and the route of administration. The severity ofthe disorder can, for example, be evaluated, in part, by standardprognostic evaluation methods. The dose and dose frequency, can varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above can be used inveterinary medicine.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the presentinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions relevant to the present invention, inparticular, those formulated as solutions, can be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds relevant to the present invention to be formulatedas tablets, pills, capsules, liquids, gels, syrups, slurries, tablets,dragees, solutions, suspensions and the like, for oral ingestion by apatient to be treated.

The therapeutic agent can be prepared in a depot form to allow forrelease into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of therapeutic agents can be, forexample, an implantable composition containing the therapeutic agent anda porous or non-porous material, such as a polymer, wherein thetherapeutic agent is encapsulated by or diffused throughout the materialand/or degradation of the non-porous material. The depot is thenimplanted into the desired location within the body and the therapeuticagent is released from the implant at a predetermined rate.

The therapeutic agent that is used in the present invention can beformed as a composition, such as a pharmaceutical composition containinga carrier and a therapeutic compound. Pharmaceutical compositionscontaining the therapeutic agent can include more than one therapeuticagent. The pharmaceutical composition can alternatively contain atherapeutic agent in combination with other pharmaceutically activeagents or drugs.

The carrier can be any suitable carrier. For example, the carrier can bea pharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used withconsideration of chemico-physical considerations, such as solubility andlack of reactivity with the active compound(s), and by the route ofadministration. In addition to, or in the alternative to, the followingdescribed pharmaceutical compositions, the therapeutic compounds of thepresent inventive methods can be formulated as inclusion complexes, suchas cyclodextrin inclusion complexes, or liposomes.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents; are well-known to thoseskilled in the art and are readily available to the public. Thepharmaceutically acceptable carrier can be chemically inert to theactive agent(s) and one which has no detrimental side effects ortoxicity under the conditions of use. The choice of carrier can bedetermined in part by the particular therapeutic agent, as well as bythe particular method used to administer the therapeutic compound. Thereare a variety of suitable formulations of the pharmaceutical compositionof the present invention. The following formulations for oral, aerosol,parenteral, subcutaneous, transdermal, transmucosal, intestinal,intramedullary injections, direct intraventricular, intravenous,intranasal, intraocular, intramuscular, intraarterial, intrathecal,intraperitoneal, rectal, and vaginal administration are exemplary andare in no way limiting. More than one route can be used to administerthe therapeutic agent, and in some instances, a particular route canprovide a more immediate and more effective response than another route.Depending on the specific disorder being treated, such agents can beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990).

Formulations suitable for oral administration can include (a) liquidsolutions, such as an effective amount of the inhibitor dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations can include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant. Capsule forms can be of the ordinary hard or soft shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and other pharmacologically compatible excipients. Lozenge formscan contain the inhibitor in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles containing the inhibitor in an inertbase, such as gelatin and glycerin, or sucrose and acacia, emulsions,gels, and the like containing, in addition to, such excipients as areknown in the art.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers can be added.

The therapeutic agent, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as pharmaceuticalsfor non-pressurized preparations, such as in a nebulizer or an atomizer.Such spray formulations also may be used to spray mucosa. Topicalformulations are well known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Injectable formulations are in accordance with the present invention.The parameters for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art [see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622 630(1986)]. For injection, the agents of the present invention can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For such transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Formulations suitable for parenteral administration can include aqueousand non-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The therapeutic agent can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,poly(ethyleneglycol) 400, glycerol, dimethylsulfoxide, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, oils, fatty acids, fattyacid esters or glycerides, or acetylated fatty acid glycerides with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethyl-cellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-beta-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations can contain from about 0.5% to about 25% byweight of the drug in solution. Preservatives and buffers can be used.In order to minimize or eliminate irritation at the site of injection,such compositions may contain one or more nonionic surfactants having ahydrophilic-lipophilic balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range fromabout 5% to about 15% by weight. Suitable surfactants includepolyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

The therapeutic agent can be made into suppositories by mixing with avariety of bases, such as emulsifying bases or water-soluble bases.Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents can be encapsulated into liposomes. Liposomes arespherical lipid bilayers with aqueous interiors. Molecules present in anaqueous solution at the time of liposome formation are incorporated intothe aqueous interior. The liposomal contents are both protected from theexternal microenvironment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly. Materials and methodsdescribed for one aspect of the present invention can also be employedin other aspects of the present invention. For example, a material sucha nucleic acid or antibody described for use in screening assays canalso be employed as therapeutic agents and vice versa.

The present invention includes the followingaspects/embodiments/features in any order and/or in any combination:

1. The present invention relates to an anti-human influenza virusmonoclonal antibody or an antigen-binding fragment thereof comprising aneutralization activity against a human influenza B virus, wherein themonoclonal antibody comprises a human monoclonal antibody or a humanizedmonoclonal antibody.

2. The anti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of any preceding or followingembodiment/feature/aspect, wherein the monoclonal antibody orantigen-binding fragment thereof has a neutralization activity againstat least a B/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, and aB/Mie/1/1993 strain.

3. The anti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of any preceding or followingembodiment/feature/aspect, wherein the monoclonal antibody orantigen-binding fragment thereof has a neutralization activity againstat least a B/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and a B/Victoria/2/1987 strain.

4. The anti-human influenza virus human monoclonal antibody of anypreceding or following embodiment/feature/aspect, wherein the humanmonoclonal antibody is produced by a hybridoma made by fusing aperipheral blood mononuclear cell (PBMC) from a human being having aninfluenza B virus infection with a fusion partner cell capable ofefficient cell fusion.

5. The anti-human influenza virus human monoclonal antibody of anypreceding or following embodiment/feature/aspect, wherein the influenzaB virus comprises at least one of a B/Florida/4/2006 strain, aB/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, aB/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04strain, a B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain.

6. The anti-human influenza B virus human monoclonal antibody of anypreceding or following embodiment/feature/aspect, wherein the fusionpartner cell is a SPYMEG cell.

7. The anti-human influenza B virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect comprising an IgG, a Fab, a Fab′, a F(ab′)2, ascFv, or a dsFv.

8. The anti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of any preceding or followingembodiment/feature/aspect, comprising:

a heavy chain variable region comprisinga first complementarity determining region (CDR1) having a first aminoacid sequence comprising SEQ ID NO: 1, 7, or 13,a second complementarity determining region (CDR2) having a second aminoacid sequence comprising SEQ ID NO: 2, 8, or 14, anda third complementarity determining region (CDR3) having a third aminoacid sequence comprising SEQ ID NO: 3, 9, or 15, anda light chain variable region comprisinga first complementarity determining region (CDR1) having a fourth aminoacid sequence comprising SEQ ID NO: 4, 10, or 16;a second complementarity determining region (CDR2) having a fifth aminoacid sequence comprising SEQ ID NO: 5, 11, or 17, anda third complementarity determining region (CDR3) having a sixth aminoacid sequence comprising SEQ ID NO: 6, 12, or 18.

9. The anti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of any preceding or followingembodiment/feature/aspect, wherein the first amino acid sequencecomprises SEQ ID NO: 1, the second amino acid sequence comprises SEQ IDNO: 2, the third amino acid sequence comprises SEQ ID NO: 3, the fourthamino acid sequence comprises SEQ ID NO: 4, the fifth amino acidsequence comprises SEQ ID NO: 5, and the sixth amino acid sequencecomprises SEQ ID NO: 6.

10. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect, wherein the anti-human influenza virusmonoclonal antibody comprises antibody 5A7.

11. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect, wherein the first amino acid sequencecomprises SEQ ID NO: 7, the second amino acid sequence comprises SEQ IDNO: 8, the third amino acid sequence comprises SEQ ID NO: 9, the fourthamino acid sequence comprises SEQ ID NO: 10, the fifth amino acidsequence comprises SEQ ID NO: 11, and the sixth amino acid sequencecomprises SEQ ID NO: 12.

12. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect, wherein the anti-human influenza virusmonoclonal antibody comprises antibody 3A2.

13. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect, wherein the first amino acid sequencecomprises SEQ ID NO: 13, the second amino acid sequence comprises SEQ IDNO: 14, the third amino acid sequence comprises SEQ ID NO: 15, thefourth amino acid sequence comprises SEQ ID NO: 16, the fifth amino acidsequence comprises SEQ ID NO: 17, and the sixth amino acid sequencecomprises SEQ ID NO: 18.

14. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect, wherein the anti-human influenza virusmonoclonal antibody comprises antibody 10C4.

15. A pharmaceutical composition comprising the anti-human influenzavirus human monoclonal antibody or antigen-binding fragment thereof ofany preceding or following embodiment/feature/aspect and apharmacologically acceptable carrier.

16. A kit for at least one of the prevention, the treatment, and thedetection of human influenza B in a human subject comprising theanti-human influenza virus human monoclonal antibody or antigen-bindingfragment thereof of any preceding or followingembodiment/feature/aspect.

17. A method of inhibiting or treating a human influenza B infection ina human subject comprising administering a therapeutically effectiveamount of the anti-human influenza B virus monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect to the human subject.

18. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, further comprising diagnosing the patientwith an influenza B infection.

19. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, further comprising monitoring for a decreasein at least one symptom of an influenza B infection.

20. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, wherein the at least one symptom comprisesfever, headache, fatigue, chills, malaise, myalgia, arthralgia, nasalcongestion, sore throat, cough, respiratory distress, or stomach pain,or any combination thereof.

21. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, wherein the anti-human influenza virus humanmonoclonal antibody or antigen-binding fragment thereof of claim 1 isadministered in combination with one or more additional therapiesdirected to influenza B.

22. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, wherein the combination acts synergisticallyto inhibit or treat the influenza B infection.

23. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, wherein the one or more additional therapiescomprise a neuraminidase inhibitor, a hemagglutinin inhibitor, ananti-inflammatory agent, or any combination thereof.

24. The method of inhibiting or treating a human influenza B infectionin a human subject of any preceding or followingembodiment/feature/aspect, wherein the neuraminidase inhibitor compriseszanamivir, oseltamivir, peramivir, laninamivir, any pharmaceuticallyacceptable salt thereof, or any combination thereof.

25. Use of the anti-human influenza B virus human monoclonal antibody orantigen-binding fragment thereof of any preceding or followingembodiment/feature/aspect to manufacture a medicament for inhibiting ortreating a human influenza B infection in a human subject.

26. A method of detecting human influenza B in a human subjectcomprising; contacting a sample from the human subject with theanti-human influenza B virus antibody or antigen-binding fragmentthereof of any preceding or following embodiment/feature/aspect; and

detecting the presence or absence of a human influenza B virus in thehuman subject based on whether the antibody binds a hemagglutinin (HA)protein of the human influenza B virus.

27. A method for producing an anti-human influenza B virus humanmonoclonal antibody comprising:

producing a hybridoma by fusing a peripheral blood mononuclear cell(PBMC) from a human being having an influenza B virus infection with afusion partner cell capable of efficient cell fusion; andobtaining an anti-human influenza virus monoclonal antibody from thehybridoma.

28. The method for producing an anti-human influenza B virus humanmonoclonal antibody of any preceding or followingembodiment/feature/aspect, wherein the influenza B virus comprises atleast one of a B/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and B/Victoria/2/1987 strain.

29. The method for producing an anti-human influenza virus humanmonoclonal antibody of any preceding or followingembodiment/feature/aspect, wherein the fusion partner cell is a SPYMEGcell.

30. An anti-human influenza virus human monoclonal antibody produced bythe method of any preceding or following embodiment/feature/aspect.

The present invention can include any combination of these variousfeatures or embodiments above and/or below as set forth in sentencesand/or paragraphs. Any combination of disclosed features herein isconsidered part of the present invention and no limitation is intendedwith respect to combinable features.

The present invention will be further clarified by the followingexamples, which are intended to be exemplary of, but not limiting, thepresent invention. Human materials were collected using protocolsapproved by the Institutional Review Boards of the Research Institutefor Microbial Diseases, Osaka University (#19-8-6). Animal studies wereconducted under the applicable laws and guidelines for the care and useof laboratory animals in the Research Institute for Microbial Diseases,Osaka University. They were approved by the Animal Experiment Committeeof the Research Institute for Microbial Diseases, Osaka University(#H21-24-0), as specified in the Fundamental Guidelines for the ProperConduct of Animal Experiment and Related Activities in Academic ResearchInstitutions under the jurisdiction of the Ministry of Education,Culture, Sports, Science and Technology, Japan, 2006. The NationalInstitute of Infectious Diseases and Dr. Shin-ichi Tamura (the NationalInstitute of Infectious Diseases) provided viral strains. NatsukoFukura, Azusa Asai, Tadahiro Sasaki, and Yohei Watanabe provided helpfuladvice and technical assistance. Data are expressed as the means+ or−standard errors of the means (SEM). Statistical analysis was performedby Student's t test. A P value of <0.05 was considered significant.

Eight influenza B vaccine strains (B/Victoria/2/1987, B/Mie/1/1993,B/Shandong/7/1997, B/Yamanashi/166/1998, B/Johannesburg/5/1999,B/Shanghai/361/2002, B/Malaysia/2506/2004, and B/Florida/4/2006) and themouse-adapted strain B/Ibaraki/2/1985 were used. TheB/Malaysia/2506/2004 and B/Florida/4/2006 strains were kindly providedby the National Institute of Infectious Diseases, Tokyo, Japan.Mouse-adapted B/Ibaraki/2/1985 strain was provided by Dr. S. Tamura,National Institute of Infectious Diseases (Chen et al., Vaccine19:1446-1455). Viruses were propagated either in Madin-Darby caninekidney (MDCK) cells or in 9-day-old embryonated chicken eggs.Infectivity was titrated by focus-forming assay.

EXAMPLES

The present invention will be further clarified by the followingexamples, which are intended to be exemplary of, but not limiting, thepresent invention.

Example 1

Human monoclonal antibodies (HuMAbs) were prepared in accordance withthe procedure described in Kubota-Koketsu et al., Biochemical andBiophysical Research Communications 387:180-185 (2009). Healthyvolunteers were vaccinated with the HA split vaccine includingA/Brisbane/59/2007 (H1N1), A/Uruguay/716/2007 (H3N2), andB/Florida/4/2006 strains. One to two weeks later, the vaccine-derivedPBMCs were fused with SPYMEG cells and after screening and cloning,three hybridoma clones producing HuMAbs, designated 5A7, 3A2, and 10C4,were established. The reactivity of the HuMAbs was tested by IFA andWestern blotting.

Briefly, 10 ml blood was drawn from a healthy volunteer vaccinated inthe 2008/2009 winter season with trivalent HA split vaccine, whichincluded A/Brisbane/59/2007, A/Uruguay/716/2007, and B/Florida/4/2006(The Research Foundation for Microbial Diseases of Osaka University,Kagawa, Japan), and then the PBMCs were collected by density gradientcentrifugation through Ficoll-Paque Plus (GE Healthcare, Uppsala,Sweden). SPYMEG cells, established from mouse myeloma cell lineSP2/0-Ag14 and human megakaryoblastic cell line MEG-01, were used asfusion partner cells. SPYMEG cells are non-secretors of human and murineimmunoglobulin. The PBMCs were fused with SPYMEG cells usingpolyethylene glycol #1500 (Roche Diagnostics, Mannheim, Germany). Thefused cells were cultured in Dulbecco's modified Eagle medium (DMEM;Invitrogen, Carlsbad, Calif.) supplemented with 15% fetal bovine serumand hypoxanthine-aminopterin-thymidine. The first screening for MAbspecific for influenza viruses was performed by immunofluorescence assay(IFA). For the IFA, the infected cells were fixed with absolute ethanoland then reacted with hybridoma supernatant for 30 min at 37 deg C.,followed by incubation with FITC-conjugated anti-human IgG for 45 min at37 deg C. The cells in the specific MAb-positive wells were cloned bylimiting dilution, then followed by a second screening by IFA. Hybridomacells taken from IFA-positive wells that had a single colony per wellwere cultured and expanded in Hybridoma-SFM (Invitrogen). MAb waspurified from 100 ml hybridoma culture supernatant by affinitychromatography using HiTrap Protein G HP Columns (GE Healthcare) andthen dialyzed into phosphate buffered saline (PBS) using Slide-A-Lyzer®Dialysis Cassettes (Thermo Scientific, Waltham, Mass.).

For IgG isotyping, ELISA microplates (Maxsorp; Nunc, Copenhagen,Denmark) were coated overnight at 4 deg C. with goat anti-human IgG(Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa.) in 0.05 Msodium bicarbonate buffer (pH 8.6). After washing with PBS including0.1% Tween-20, the wells were blocked with 0.5% BSA in PBS for 1 hour at37 deg C. After washing again, the wells were incubated with hybridomasupernatants or control serum for 2 hours at 37 deg C. Following furtherwashing, the wells were incubated with HRP-conjugated anti-human IgG1,IgG2, IgG3, or IgG4 (SouthernBiotech, Birmingham, Ala.) for 1 hour at 37deg C. The wells were washed five times followed by incubation with TMBperoxidase substrate (KPL, Gaithersburg, Md.) at room temperature in thedark. After 20 minutes, the reaction was stopped with 2N H2SO4 solution.The color development was read at 450 nm in an ELISA Photometer (BiotekELISA Reader; Biotek, Winooski, Vt.). All samples were run intriplicate.

For the sequencing of HuMAb variable regions, total RNA extracted fromthe hybridoma using an RNeasy Mini Kit (Qiagen) was subjected to RT-PCRusing a PrimeScript RT reagent Kit (Takara, Shiga, Japan) with an oligo(dT) primer. The coding region of the H- and L-chains of HuMAb wasamplified by PCR with the following primers:5′-ATGGAGTTTGGGCTGAGCTGGGTT-3′ (H-chain-forward) (SEQ ID NO. 19) and5′-CTCCCGCGGCTTTGTCTTGGCATTA-3′ (H-chain-reverse) (SEQ ID NO. 20); and5′-ATGGCCTGGRYCYCMYTCYWCCTM-3′ (L-chain-forward) (SEQ ID NO. 21) and5′-TGGCAGCTGTAGCTTCTGTGGGACT-3′ (L-chain-reverse) (SEQ ID NO. 22). PCRproducts were ligated into pGEM-T Easy Vector (Promega) and theirsequences were analyzed using a BigDye Terminator v3.1 Cycle SequencingKit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, FosterCity, Calif.).

IgG plasmids were constructed using T Easy Vectors with the variableregion gene of H- and L-chains were subjected to PCR to add restrictionenzyme sites and a Kozak sequence with the following primer sets(restriction enzyme sites are underlined):5′-ATTTGCGGCCGCCATGGAGTTTGGGCTGAG-3′ (HC_(—)5Fw_NotI; H-chain-forward)(SEQ ID NO. 23) and 5′-ATACTCGAGGGTGCCAGGGGGAAGACCGATG-3′(HC_Reverse_XhoI; H-chain-reverse) (SEQ ID NO. 24); and5′-ATTTGCGGCCGCCATGGCCTGGGCTCTGCT-3′ (5A7Lambda_(—)18 Fw_NotI;L-chain-forward) (SEQ ID NO. 25) and5′-ATACTCGAGGGCGGGAACAGAGTGACCGTGG-3′ (Lambda_Reverse_XhoI;L-chain-reverse) (SEQ ID NO. 26). PCR products of the coding region ofH- and L-chains were digested by restriction enzymes, Not I and Xho I,and then ligated to expression vectors, pQCXIP-hCH and pQCXIH-hC lambda,which have a human immunoglobulin-constant region of gamma and lambdachains (MBL), respectively.

All of three HuMAbs reacted with the HA protein in influenza B virus(Table 1). HuMAbs, 5A7 and 10C4 were IgG1 isotype, and 3A2 was IgG3(Table 1). Sequencing analysis of the V_(H) and V_(L) region of thethree HuMAbs revealed that each had different amino acid residues inantigenic regions including the complementarity-determining regions(CDRs) (Tables 2 and 3).

TABLE 1 Table 1: Pattern of Reactivity of HuMAbs. Target Isotype IFAReducing WB 5A7 HA of influenza B IgG1 +¹ + 3A2 HA of influenza B IgG3 +− ² 10C4 HA of influenza B IgG1 + − ¹Positive result ² Negative result

TABLE2 Table 2: Deduced Amino Acid Sequences of CDRs inthe V_(H) of three HuMAbs. SEQ. SEQ. SEQ. ID ID ID V_(H) CDR1 No. CDR2No. CDR3 No. 5A7 NYGMH 1 VVWYDGLIKY 2 DLQPPHSPYGM 3 YADSVKG DV 3A2 SYYWS7 YVYNSGSTR 8 APDDYYDSVGYY 9 YNPSLKS YGCPYFDS 10C4 NYAMS 13 AISGGGDWT 14DVTYLYDSSGYY 15 YYADSVKG YGGADRDYYFDY

TABLE3 Table 3: Deduced Amino Acid Sequences of CDRsin the Y_(L) of three HuMAbs. SEQ. SEQ. SEQ. ID ID ID V_(L) CDR1 No.CDR2 No. CDR3 No. 5A7 SGSSSNI 4 NNNQRPS 5 AAWDDSLTVS 6 GSNDVY 3A2RASPSIA 10 GASTRAT 11 QQYSNWPRT 12 DNLA 10C4 SGGSSNI 16 SNNQRPL 17QQWDDSLNGWV 18 GSNYVN

The neutralizing activities of HuMAbs were determined as follows. Thevirus neutralization (VN) assay was carried out in accordance with Okunoet al. 28:1308-1313 (1990), with minor modification. MAb at aconcentration of 100 mcg/ml was serially diluted four-fold with MinimumEssential Medium (MEM; Invitrogen) and incubated with 200 focus-formingunits (FFU) of viruses at 37 C. for 1 hour. Then, MDCK cells wereadsorbed with the mixtures at 37 C. for 1 hour. After incubation for 12hours, the cells were fixed and subjected to IFA. The lowestconcentration of MAb that inhibited 50% of viral growth was designatedthe VN₅₀ titer. In the focus formation assay, MDCK cells in a 96-wellplate were adsorbed with viruses diluted serially 10-fold at 37 deg C.for 1 hour. The cells were then washed with PBS and incubated at 37 degC. for 12 hours. The cells were fixed and subjected to IFA.

VN assays were performed with the three HuMAbs. HuMAb 5A7 had a lowerVN₅₀ (6.25 to 25 mcg/ml) compared with 3A2 and 10C4; however, 5A7neutralized the Yamagata and Victoria lineages isolated during 1985 to2006. HuMAbs 3A2 and 10C4 had a VN₅₀ of 0.02 to 6.25 mcg/ml for Yamagatalineage, whereas they hardly neutralized any Victoria lineage exceptmouse-adapted B/Ibaraki/2/1985, which was neutralized slightly by 3A2(Table 4). To clarify the mechanism of neutralization by the threeHuMAbs, HI and fusion inhibition assays were also performed. In thehemagglutinin inhibition (HI) assay, viral titers were determined with ahemagglutination assay. Briefly, the viruses were serially dilutedtwo-fold with PBS and mixed with 0.7% (v/v) human O-type red bloodcells. After incubation at room temperature for 1 hour, hemagglutinationunits (HAUs) were estimated. Next, HI titration was performed asfollows. MAb at a concentration of 100 mcg/ml was serially dilutedtwo-fold and mixed with 8 HAU per 50 l of viral sample. After incubationat 37 C. for 1 hour, the mixtures were further incubated with 0.7% (v/v)human red blood cells for 1 hour at room temperature. The lowestconcentration of MAb that completely inhibited hemagglutination wasdesignated the HI titer.

For the fusion inhibition assay, cell-cell fusion was accomplished asdescribed previously (Okuno et al., Journal of Virology 67:2552-2558).Briefly, monkey kidney cell line CV-1 cells were infected withB/Florida/4/2006 at an MOI of 0.3. After incubation for 24 hours, thecells were washed with MEM and then incubated for 15 min at 37 deg C. inMEM supplemented with 2.5 mcg/ml of acetylated trypsin (Sigma, St.Louis, Mo.). After washing, the cells were incubated for 30 min withdiluted HuMAbs. Thereafter, the cells were treated for 2 min at 37 degC. with MEM supplemented with 10 mM MES and 10 mM HEPES (pH 5.5). Afterthe medium was completely removed by washing, the cells were incubatedfor 3 hours. Then they were fixed with absolute methanol and stainedwith Giemsa (Wako, Osaka, Japan).

TABLE 4 Table 4. Characterization of HuMAbs HuMAb 5A7 3A2 10C4 VN₅₀(μg/ml)¹ Yamagata lineage B/Florida/4/2006 6.25 0.10 0.39B/Shanghai/361/2002 6.25 6.25 0.39 R/Johannesburg/5/1999 6.25 0.10 0.39B/Yamanashi/166/1998 6.25 0.10 0.39 B/Mie/1/1993 6.25 0.10 0.39 Victorialineage B/Malaysia/2506/2004 6.25 >100 >100 B/Shandong/7/199725 >100 >100 B/Victria/2/1987 25 >100 >100 Mouse-adaptedB/Ibaraki/2/1985 25 100 >100 HI (μg/ml)² B/Florida/4/2006 25 0.39 0.39Fusion inhibition (μg/ml)³ B/Florida/4/2006 100 25 25 ¹The results areshown as the lowest concentrations of purified HuMAbs that inhibited 50%of viral growth in vitro. ²The results are shown as the lowestconcentrations of purified HuMAbs that completely inhibitedhemagglulination. ³The results are shown as the lowest concentrations ofpurified HuMAbs that showed cell fusion inhibition.

Accordingly, all three HuMAbs had HI activity and also inhibitedcell-cell fusion (Table 4). However, 3A2 and 10C4 showed markedly higherHI titers (0.39 mcg/ml) than 5A7 (25 mcg/ml). These results indicatethat all three HuMAbs should inhibit viral binding to the cell membrane.

Example 2

To determine the epitope regions of B/Florida/4/2006 recognized by thethree HuMAbs, escape mutants were selected. The escape mutants wereselected by the incubation of B/Florida/4/2006 with HuMAbs. Escapemutants were selected by culturing B/Florida/4/2006 in the presence ofMAb as described previously (Gulati et al., Journal of Virology76:12274-12280), with minor modification. Viruses were incubated withMAb serially diluted 10-fold (to give final concentrations of 0.0025 to2.5 mcg/ml) at 37 C. for 1 hour. Then MDCK cells were inoculated withthe mixtures and cultured in DMEM/F-12+GlutaMAX™-I supplemented with0.4% BSA, antibiotics, and 2 mcg/ml acetylated trypsin. After culturingfor 72 hours, the supernatants were collected and subjected to VN and HIassays. Those viral samples that showed a reduced VN₅₀ and HI titer weresubjected to direct sequencing analysis of the entire HA gene.

Direct sequencing analysis was performed as follows. Viral RNA extractedwith QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was subjectedto one step RT-PCR (Superscript™ III One-Step RT-PCR System withPlatinum® Taq High Fidelity; Invitrogen) with the following HA primerset: 5′-CAGAATTCATGAAGGCAATAATTGTACTAC-3′ forward (SEQ ID NO. 27) and5′-CTCCGCGGCCGCTTATAGACAGATGGAGCATGAAACG-3′ reverse (SEQ ID NO: 28). ThePCR products were purified with Qiaquick PCR Purification Kit (Qiagen).After electrophoresis, the discrete band was extracted using theQiaquick Gel Extraction Kit (Qiagen) and sequenced.

HA plasmids were constructed as follows. The HA gene of B/Florida/4/2006was amplified by one step RT-PCR and inserted into the pGEM-T EasyVector (Promega, Madison, Wis.). Mutant and truncated HA genes weregenerated by site-directed mutagenic PCR (GeneTailor™ Site-DirectedMutagenesis System; Invitrogen) and conventional PCR (Expand HighFidelity^(PLUS) PCR System; Roche), respectively, using the HA plasmidof B/Florida/4/2006 inserted into pGEM-T easy vector. Each of theplasmids was subcloned into the expression vector pCAGGS/MCSII (Ueda etal., Journal of Virology 84: 3068-3078). The expression plasmids weretransfected into human embryonic kidney 293T cells withlipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions.

The amino acid sequences of the HA protein in the escape mutants werecompared with the original B/Florida/4/2006. Asterisks in FIG. 1indicate the amino acid residues different between the original virusand the escape mutants. Interestingly, each escape mutant of 3A2 and10C4 had amino acid substitutions at identical positions 194D and 196T;amino acid numbering was started after the signal peptide (Wang et al.,Journal of Virology 82:3011-3020). These positions are located at the190-helix antigenic site near the receptor-binding site (Wang et al.,Journal of Virology 82:3011-3020). Remarkably, in the presence ofserially diluted 5A7, escape mutants were not established even after thevirus was passaged ten times, implying that the amino acid sequencerecognized by 5A7 was essential for viral survival. HuMAb 5A7 reacted tothe HA0 protein by Western blotting under reducing conditions,suggesting that 5A7 had a sequential epitope. Thus, the epitope regionof 5A7 was further investigated using HA truncation vectors containingHA segments of varying length. Western blotting with 5A7 was carried outon 293T cells transfected with the truncated HA expression vectors.HuMAb 5A7 reacted with truncated HA segments that included amino acidresidues 1 to 324 but not to those with residues 1 to 314 (FIG. 2).These results indicate that 5A7 recognizes amino acid residues between315 to 324, IGNCPIWVKT (SEQ. ID NO. 44) in the HA protein, which locatesnear the C terminal of the HA 1 protein. Notably, this region is ahighly conserved domain in influenza B viruses.

Example 3

HuMAb 3A2 showed low reactivity against B/Shanghai/361/2002 and wastherefore examined for an additional distinct epitope region. To dothis, various chimeric sequences of HA were constructed fromB/Florida/4/2006 and B/Shanghai/361/2002, which differ at seven residues(positions 37, 40, 88, 131, 227, 249, 456), expressed in plasmids, andtransfected into 293T cells. IFA of the chimeric HA proteins expressedin 293T cells showed that 131P and 227S were essential for the reactionwith 3A2 (FIG. 3). These results indicate that the epitope of 3A2 isdependent on residues at positions 131, 194, 196, and 227, and theepitope of 10C4 is dependent on residues at positions 194 and 196. Theepitope regions to which the three HuMAbs map are shown in a HA trimerthree-dimensional model in FIG. 4. HuMAbs 3A2 and 10C4 recognized thetop of the globular head including the 190-helix antigenic site, whereas5A7 reacted with the stalk region distant from the viral membrane.

HuMAb, 5A7 recognizes the stalk region of the HA protein, as do almostall broadly neutralizing MAbs for influenza A viruses. The amino acidsequence in the stalk region is highly conserved, implying that theamino acid residues would not easily mutate. Indeed, the amino acidresidues in the epitope region did not mutate even when the virus waspassaged ten times under 5A7-treatment conditions, whereas mutantsdeveloped quickly in the presence of 3A2 or 10C4. Failure to establishescape mutants in the presence of 5A7 is an advantage for this HuMAb asa therapeutic candidate. Although 5A7 reacted with both of Yamagata andVictoria lineages, the concentration required for VN₅₀ was higher thanthose of 3A2 and 10C4. Such results can be explained by either adifference in binding affinity or in physical accessibility of HuMAbs tothe epitope region. Although binding affinity was not examined in thisstudy, accessibility can be estimated using epitope mapping. HuMAbs 3A2and 10C4 recognized the 190-helix site distant from the viral membrane,whereas the epitope region of 5A7 localized to the stalk region,indicating that 5A7 would have more difficulty physically accessing theHA protein. Modification of the HuMAb structure and improvement ofbinding affinity should lead to the development of better therapeuticantibodies.

MAbs recognizing the globular head show strong HI activity, whereasthose against the stalk region usually do not show any. Thus, it isconsidered that MAbs against the globular head inhibit the receptorbinding step and that MAbs against the stalk region inhibit the fusionstep in viral replication. The HuMAbs that recognize the 190-helix inthe globular head near the receptor binding site, 3A2 and 10C4, did infact have strong HI activities, suggesting that these HuMAbs inhibitedbinding to the receptor. Surprisingly, 5A7 also showed weak HI activity,implying that 5A7 would also inhibit the binding step. It would beattributed that 5A7 recognizes the stalk region distal from the viralmembrane, whereas MAbs that do not show HI activity recognize a moreproximal position of the stalk. All three HuMAbs also showed fusioninhibition activity. HuMAbs bound to the HA protein could secondarilydisturb the structural change of HA depending on low pH.

Example 4

The activity of 5A7 as a passive transfer therapy in influenza B viralinfection was examined in mice. In the passive transfer experiments,before infection, mice were anesthetized by intraperitonealadministration of pentobarbital sodium (Somnopentyl; Kyoritsu SeiyakuCorporation, Tokyo, Japan). Six-week old female BALB/c mice from JapanSLC Inc. were used. Mice were given 5, 10, or 15 mg/kg HuMAbintraperitoneally at 4, 24, 48, or 72 hours after challenge with 25 mcl(microlitre) mouse-adapted B/Ibaraki/2/1985 virus at a lethal dose(2.5×10⁴ FFU/mouse). Mice were weighed daily and sacrificed if they fellto 60% of starting weight. To titrate the viruses in the infected lungs,B/Ibaraki/2/1985 or B/Florida/4/2006 were infected at 2.5×10⁴ or 5.0×10³FFU/mouse, respectively. The lungs were harvested 3 and 6 dayspost-infection and virus titers in lung homogenates were determined byfocus-forming assay.

Mice were treated intraperitoneally with 5, 10, or 15 mg/kg 5A7 at 4hours post-challenge with a lethal dose of mouse-adaptedB/Ibaraki/2/1985. Survival rate and weight change were checked daily andwhen the body weight had dropped to less than 60% of starting weight,mice were sacrificed. Complete therapeutic efficacy against the viruswas seen with each dose of 5A7 tested (FIG. 5). The weight change wasparticularly mild in the groups treated with 5A7 at 10 or 15 mg/kg (FIG.6). In mice treated with 10 mg/kg 5A7, the viral load in the lungs wastitrated three and six days post-infection with mouse-adaptedB/Ibaraki/2/1985 or B/Florida/4/2006. The viral titers weresignificantly lower in 5A7-treated mice compared to untreated controlsfor both viral infections (FIG. 7). Finally, mice were given 5A7 at 10mg/kg intraperitoneally at 4, 24, 48, or 72 hours post-challenge with alethal dose of mouse-adapted B/Ibaraki/2/1985, and the survival rate andweight change were monitored. When injected four hours post-infection,5A7 treatment showed complete therapeutic efficacy. Notably, most of themice were alive in the 5A7-treated group at 24 hours post-infection.Moreover, several mice survived even after treatment with 5A7 more than48 hours post-infection (FIGS. 8 and 9). These results indicate that 5A7has the potential for development as a therapy against influenza B viralinfection.

Mouse-adapted B/Ibaraki/2/1985 was used to examine the kinetics ofsurvival rate and weight change in the passive transfer experiments asother viral strains are not lethal to mice even if passaged severaltimes in vivo. HuMAb 5A7 protected mice against mouse-adaptedB/Ibaraki/2/1985 challenge even when administered 72 hourspost-infection, although 5A7 had shown the lowest sensitivity tomouse-adapted B/Ibaraki/2/1985 in vitro. These results suggested that5A7 would have therapeutic efficacy against a wide spectrum of influenzaB viruses, and in fact the lung viral titers of both mouse-adaptedB/Ibaraki/2/1985 and B/Florida/4/2006 were reduced significantly under5A7-treatment conditions.

Example 5

The neutralizing efficacy of CHO-K1-derived 5A7 was examined in vitro.5A7 was synthesized in CHO-K1 cells and examined for neutralizingefficacy against influenza B viruses. CHO-K1 cells were transfected withfull-length variable region genes of 5A7 in the pQC vector to establisha stable cell line secreting 5A7 (5A7/CHO-K1). For stable expressionusing mammalian cells, CHO-K1 cells were cultured in 5% CO₂ at 37 deg C.in DMEM containing 10% fetal bovine serum. Cells grown on 6-well plates(Corning, Corning, N.Y.) were used for transfection with pQCXIP-hCH andpQCXIH-hC lambda expression vectors using Lipofectamine2000 transfectionreagent (Invitrogen). Transfected cells were incubated in 5% CO₂ at 37deg C. in DMEM with 1 mcg/ml of puromycin and 100 mcg/ml of hygromycinfor 3 weeks. Cells were then replated to 15-cm dishes (Corning) andincubated, and colonies were picked and cultured as putative cell linesstably expressing IgG. The transformants at 90% confluence on 15-cmdishes had the medium changed to serum-free Nutrient Mixture F-12 Ham's(Sigma), cells were then cultured in 5% CO₂ at 37 deg C. for 1 week.Recombinant IgGs were purified from culture medium using HiTrap ProteinG HP Columns. The purified IgGs were dialyzed against PBS and thenconcentrated using an Amicon Ultra Centrifugal Filter (Millipore,Billerica, Mass.). In vitro viral neutralization tests were performedusing purified 5A7/CHO-K1, and neutralizing activity was found againstboth the viral strains examined (B/Florida/4/2006 andB/Malaysia/2506/2004). Notably, the neutralizing activity of 5A7/CHO-K1was similar to that of 5A7 produced by the hybridoma (FIG. 10).

Example 6

HuMabs 5A7, 3A2 and 10C4 were subjected to surface plasmon resonanceanalysis to examine their binding affinities. Each HuMab was immobilizedon the surface of the sensor chip. The vaccine antigen, Ha protein ofB/Florida/4/2006, at concentrations 12.5, 25, 50, 100 and 200 nM wasconsecutively injected on the chip surface and the association anddissociation phases were monitored. KD value could not be calculated for5A7 precisely as it was difficult to dissociate from HA (Table 5).

TABLE 5 Kinetic constants of HuMAbs binding to influenza B virus-derivedHA. kon¹ (S⁻¹) koff² (M⁻¹S⁻¹) K_(D) (M) 5A7 1.8 × 10³ <1.0 × 10⁻⁵  <5.6× 10⁻⁹  3A2 5.3 × 10⁴ 2.1 × 10⁻⁵  4.0 × 10⁻¹⁰ 10C4 1.6 × 10⁴ 2.8 × 10⁻⁵1.8 × 10⁻⁹ ¹Association rate constant. ²Dissociation rate constant.

Sequences of the V_(H) and V_(L) region of the three HuMAbs werecompared and analyzed to the closest germline sequences using IgBlastsoftware in NCBI database. These three HuMAbs were derived fromdifferent germ lines except D region V_(H) of 3A2 and 10C4 (FIG. 11-13).

REFERENCES

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Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

Other embodiments of the present invention will be apparent to thoseskilled in the art from consideration of the present specification andpractice of the present invention disclosed herein. It is intended thatthe present specification and examples be considered as exemplary onlywith a true scope and spirit of the invention being indicated by thefollowing claims and equivalents thereof.

1. An anti-human influenza virus monoclonal antibody or anantigen-binding fragment thereof comprising a neutralization activityagainst a human influenza B virus, wherein the monoclonal antibodycomprises a human monoclonal antibody or a humanized monoclonalantibody.
 2. The anti-human influenza virus monoclonal antibody orantigen-binding fragment thereof of claim 1, wherein the monoclonalantibody or antigen-binding fragment thereof has a neutralizationactivity against at least a B/Florida/4/2006 strain, aB/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, aB/Yamanashi/166/1998 strain, and a B/Mie/1/1993 strain.
 3. Theanti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of claim 1, wherein the monoclonal antibody orantigen-binding fragment thereof has a neutralization activity againstat least a B/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and a B/Victoria/2/1987 strain.
 4. The anti-human influenzavirus human monoclonal antibody of claim 1, wherein the human monoclonalantibody is produced by a hybridoma made by fusing a peripheral bloodmononuclear cell (PBMC) from a human being having an influenza B virusinfection with a fusion partner cell capable of efficient cell fusion.5. The anti-human influenza virus human monoclonal antibody of claim 4,wherein the influenza B virus comprises at least one of aB/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, aB/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, aB/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a B/Shandong/7/1997strain, and a B/Victoria/2/1987 strain.
 6. The anti-human influenza Bvirus human monoclonal antibody of claim 4, wherein the fusion partnercell is a SPYMEG cell.
 7. The anti-human influenza B virus monoclonalantibody or antigen-binding fragment thereof of claim 1 comprising anIgG, a Fab, a Fab′, a F(ab′)2, a scFv, or a dsFv.
 8. The anti-humaninfluenza virus monoclonal antibody or antigen-binding fragment thereofof claim 1, comprising: a heavy chain variable region comprising a firstcomplementarity determining region (CDR1) having a first amino acidsequence comprising SEQ ID NO: 1, 7, or 13, a second complementaritydetermining region (CDR2) having a second amino acid sequence comprisingSEQ ID NO: 2, 8, or 14, and a third complementarity determining region(CDR3) having a third amino acid sequence comprising SEQ ID NO: 3, 9, or15, and a light chain variable region comprising a first complementaritydetermining region (CDR1) having a fourth amino acid sequence comprisingSEQ ID NO: 4, 10, or 16; a second complementarity determining region(CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 5, 11,or 17, and a third complementarity determining region (CDR3) having asixth amino acid sequence comprising SEQ ID NO: 6, 12, or
 18. 9. Theanti-human influenza virus monoclonal antibody or antigen-bindingfragment thereof of claim 8, wherein the first amino acid sequencecomprises SEQ ID NO: 1, the second amino acid sequence comprises SEQ IDNO: 2, the third amino acid sequence comprises SEQ ID NO: 3, the fourthamino acid sequence comprises SEQ ID NO: 4, the fifth amino acidsequence comprises SEQ ID NO: 5, and the sixth amino acid sequencecomprises SEQ ID NO:
 6. 10. The anti-human influenza virus monoclonalantibody or antigen-binding fragment thereof of claim 8, wherein thefirst amino acid sequence comprises SEQ ID NO: 7, the second amino acidsequence comprises SEQ ID NO: 8, the third amino acid sequence comprisesSEQ ID NO: 9, the fourth amino acid sequence comprises SEQ ID NO: 10,the fifth amino acid sequence comprises SEQ ID NO: 11, and the sixthamino acid sequence comprises SEQ ID NO:
 12. 11. The anti-humaninfluenza virus monoclonal antibody or antigen-binding fragment thereofof claim 8, wherein the first amino acid sequence comprises SEQ ID NO:13, the second amino acid sequence comprises SEQ ID NO: 14, the thirdamino acid sequence comprises SEQ ID NO: 15, the fourth amino acidsequence comprises SEQ ID NO: 16, the fifth amino acid sequencecomprises SEQ ID NO: 17, and the sixth amino acid sequence comprises SEQID NO:
 18. 12. A pharmaceutical composition comprising the anti-humaninfluenza virus human monoclonal antibody or antigen-binding fragmentthereof of claim 1 and a pharmacologically acceptable carrier.
 13. A kitfor at least one of the prevention, the treatment, and the detection ofhuman influenza B in a human subject comprising the anti-human influenzavirus human monoclonal antibody or antigen-binding fragment thereof ofclaim
 1. 14. Use of the anti-human influenza B virus human monoclonalantibody or antigen-binding fragment thereof of claim 1 to manufacture amedicament for inhibiting or treating a human influenza B infection in ahuman subject.
 15. A method of detecting human influenza B in a humansubject comprising; contacting a sample from the human subject with theanti-human influenza B virus antibody or antigen-binding fragmentthereof of claim 1; and detecting the presence or absence of a humaninfluenza B virus in the human subject based on whether the antibodybinds a hemagglutinin (HA) protein of the human influenza B virus.
 16. Amethod for producing an anti-human influenza B virus human monoclonalantibody comprising: producing a hybridoma by fusing a peripheral bloodmononuclear cell (PBMC) from a human being in an influenza B virusinfection with a fusion partner cell capable of efficient cell fusion;and obtaining an anti-human influenza virus monoclonal antibody from thehybridoma.
 17. The method for producing an anti-human influenza B virushuman monoclonal antibody of claim 16, wherein the influenza B viruscomprises at least one of a B/Florida/4/2006 strain, aB/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, aB/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04strain, a B/Shandong/7/1997 strain, and B/Victoria/2/1987 strain. 18.The method for producing an anti-human influenza virus human monoclonalantibody of claim 16, wherein the fusion partner cell is a SPYMEG cell.19. An anti-human influenza virus human monoclonal antibody produced bythe method of claim 16.